We have evidence that cardioprotective drugs and ischemic preconditioning stimulate autophagy and that inhibition of autophagy blocks cardioprotection. We suggest that autophagy is the final common pathway for many cardioprotective conditioning stimuli. We propose a novel hypothesis that autophagy is protective because it supports glutathione biosynthesis and/or amino acid transport across the autophagosomal membrane. Also, recognizing that an elevated NADH/NAD+ ratio results in ROS production from Complex I, and leads to mitochondrial damage and permeability transition pore opening, we hypothesize that interventions which shift the NADH/NAD+ ratio towards oxidation, such as pyruvate or Tat-Ndi1 administration, will decrease ROS production, preserve mitochondrial integrity, and decrease the oxidation of glutathione. Thus, we propose that a combination of agents that increase autophagy and modulate the NADH/NAD+ ratio will provide maximal cardioprotection. This combination will consist of an agent that briskly induces autophagosomal formation and an agent(s) that metabolically protects mitochondria. We will perform mechanistic studies using a rat heart Langendorff model of both stunning and necrosis. Translational studies will be evaluated in clinically relevant in situ myocardial stunning and infarction porcine preparations. We propose four specific aims: 1) Demonstrate that autophagy is necessary and sufficient for cardioprotection by conditioning agents in the rat Langendorff model using Tat-Atg5K130R. 2) Determine whether autophagy supports glutathione biosynthesis and/or proton pumping in pharmacologically conditioned rat hearts subjected to stunning and necrosis. 3) Modulate mitochondrial NADH levels to achieve cardioprotection using pyruvate or Tat-Ndi1 in rat hearts using stunning and necrosis models. 4) Optimize the upregulation of autophagy and maximize glutathione levels with preconditioning agents and pyruvate or Tat-Ndi1 to reduce myocardial stunning and infarct size in clinically relevant in situ porcine preparations. These studies will establish the fundamental mechanisms involved in autophagy and pharmacological conditioning and metabolic interventions. This knowledge will enable us to optimize cardioprotective protocols in humans. Public Health Relevance: Myocardial stunning and infarction are major short and long term causes of morbidity and mortality after percutaneous coronary interventions (PCI), reparative heart surgery, and heart transplantation. The objective of this project is to develop new therapies to increase the heart's tolerance to ischemia based on the process of autophagy. This will entail focusing on recent findings which implicate autophagy as a final common pathway for many pharmacological agents known to mimic the phenomenon of ischemic preconditioning.